1. Field of the Invention
This invention relates to a special use of a live vaccine, to new live vaccines not having been used before, to a method of producing such vaccines as well as to suitable vaccine strains, especially salmonella.
2. Description of the Related Art
The majority of salmonella-conditioned gastroenteritis infections of humans are caused by contaminated animal products. Especially chicken and chicken eggs, respectively, being infected with the at present predominantly occurring serovar Salmonella enteritis have increasingly been causing infections, recently. Nevertheless, generally all food stuffs are affected which originate from animals kept in mass-rearing. Here, normally many animals are kept in confined space, promoting the spread of infections among the animal stock.
The risk of a transmission of the infection from the infected animal to humans can be reduced by customary veterinary medical measures for the interruption of infection chains. Furthermore, thorough compliance with kitchen hygiene regulations during the processing of contaminated animal products can prevent a transmission to humans. However, especially the latter regulations are not always being considered during the storage and processing of food. Therefore, it is imperative to rule out the possibility of infected animals being processed right from the beginning. This can be achieved e.g. through a vaccination of the animal stock against salmonella infections.
Suitable salmonella live vaccines have to comply with various different conditions:
1. The virulence of the vaccine strains used in the production of the vaccines has to be adjusted in a way that guarantees a non-apparent infection on the one hand, and a sufficient persistence of the vaccine strains in the host tissue on the other hand, as a prerequisite for high immunogenicity.
2. Furthermore, the stability of the vaccine strains used with respect to their virulence and their protective properties has to be widely assured, i.e., it has to be assured that they do not mutate back into the virulent wild strain.
3. To allow for the reduction of the probability of infections it should be ascertained that the vaccine strains are not permanently being excreted alive and that they can only service for a short period of time in the environment, respectively.
The above-mentioned three conditions, which a live vaccine has to comply with, are to be discussed in detail in the following. As described in 1, the production of a suitable salmonella live vaccine is based on a reduction of the virulence (attenuation) of the pathogenic salmonella and simultaneous preservation of their antigen structures, and thus, the immunogenic effect in the host. One possibility is e.g., to employ deletion mutants, e. g. pur or aro auxotrophic clones, as vaccine strains. The attenuation level of these vaccine strains depends upon the lack of metabolites in vivo, which possibly impedes an accurate adaptation to the host to be immunized. In this respect, it is referred to the EP 0 263 528 in which stable asp mutants of Salmonella typhimurium with different virulence reduction levels are described. Vaccine strains with attenuation levels adapted to each of the different host species can be produced by selecting suitable asp mutants.
A further possibility for an attenuation consists of the employment of vaccine strains, the virulence reduction of which can be traced back to a metabolism drift mutation (called stwd mutation or marker in the following). The term "metabolism drift" comprises all essential enzymes and functionally important cell compartments, respectively, having been functionally altered by mutations, as e. g. ribosome proteins, gyrase, RNA polymerase, permease, wherein, as a result of these mutations, translation, DNA replication, DNA transcription or permeation are more or less distinctly disturbed. Such stwd mutants, furthermore, show a resistance with respect to specific antibiotics and other substances (noxious substances). Stwd mutants can easily be obtained in laboratories as chromosomal antibiotic resistance mutants. In this respect, from the EP 0 263 528 e. g. stwd mutants with a resistance against nalidixic acid (Nal), streptomycin (Sm) or rifampicin (Rif) are known. Especially if several stwd markers are incorporated into one vaccine strain (double or triple marker vaccine strain), de facto unlimited possibilities are obtained for the production of a desired attenuation level adapted to suit every specific host species.
With respect to the prior art "attenuation by means of stwd mutations" it is referred to the following publications: DD-WP 155 294; DD-WP 218 834; DD-WP 235 828; DD-WP 253 182; DD-NP 253 184; DD-WP 281 118; DD-WP 294 420; EP 0 263 528.
A further (mentioned above under 2) condition is that the attenuated vaccine strains obtained by mutation do not mutate back into the virulent wild strain. The required stability can, on the one hand, be achieved by only employing vaccine strains with which no reversions can be detected in vitro or whose reversion ratios are &lt;10.sup.7. A further possibility is to employ vaccine strains comprising several mutations which independently reduce virulence. Here, the probability of a back mutation can almost be excluded.
The final condition, mentioned above under 3 in connection with the term "interruption of infection chains", especially concerns the risks with respect to a possible excretion and permanent survival of the vaccine strains outside the vaccinated host. In this respect, it is desirable to reduce the excretion and the capability of survival of the vaccine strains in the environment. To guarantee a sufficient immune response, on the other hand, the capability of temporary survival of the vaccine strains in the host tissue after e. g. oral or parenteral application should only be slightly impaired or not at all. Vaccine strain mutants complying with such requirements are known e. g. from the DD-WP 218 836, DD-WP 231 491, DDWP 253 182, DD-WP 253 183, DD-WP 253 184, and EP 0 263 528. In the prior art it is suggested to optimize suitable vaccine strains by employing so-called anti-epidemic markers for the reduction of excretion and the capability of survival in the environment. The term anti-epidemic marker characterizes outer envelope mutations in a broader sense, causing a functional variation of the permeability barrier in the outer membrane.
Vaccine strains can be provided with different anti-epidemic markers depending upon the intended application form. The anti-epidemic markers known at present are divided into three groups, depending on the alterations they cause in the outer membrane of the vaccine strain. The first group comprises the so-called hst markers. The incorporation of an hst marker causes the vaccine strain to become highly sensitive towards bile, anionic detergents, macrolide antibiotics and other noxious substances. Owing to the high sensitivity towards bile, there is a reduced excretion with feces caused by the inactivation of the vaccine strains already occurring in the intestinal lumen. If vaccine strain bacteria are excreted, they only have a shortened survival time in the environment, due to the lack of the permeability barrier in the outer membrane against tensides and macrolides and other noxious substances. Therefore, an infection can almost be excluded when using vaccine strains including hst markers. When employing the usual doses of vaccine, vaccine strains comprising hst markers can only be applied parenterally, however. If applied orally, due to the high sensitivity towards bile, the virulence is influenced to such an extent that a sufficient immune response can only be achieved by employing extremely high doses of vaccine. Therefore, the solution for an oral application would be to provide vaccine strains including an anti-epidemic marker from one of the other two known groups. One group comprises the so-called rbt markers (reversion to bile tolerance). The rbt marker can be obtained by mutation from the hst marker. It provides the vaccine strain with an anti-epidemic potency just as the hst marker does. However, in contrast to the hst marker the vaccine strain comprising an rbt marker is tolerant towards bile, and can therefore be applied orally without a reduction of the virulence impairing the vaccination effect. The same stands for a further group, the so-called rtt marker (reversion to tenside tolerance). The rtt marker can be obtained by mutation from the rbt marker. The vaccine strain comprising the rtt marker is tolerant towards tensides and simultaneously possesses a sufficient anti-epidemic potency due to the remaining high sensitivity towards macrolides and other noxious substances. Also the rtt marker strain can be applied orally without any problems.
Taking this information and these publications as a basis, live vaccines can be produced which comply with all conditions required. The adaptation to the respective host can be effected, e.g. in animal test series.
A further problem remains to be solved. Even the compliance with all precautions does not exclude the possibility of a person dealing e.g. with the vaccination of the animals or the production of the vaccines, coming into contact with the in fact attenuated but, nevertheless, still pathogenic salmonella vaccine strains. Where healthy people are concerned, there is hardly any risk of an infection. However, if the immune system of people is weakened (e. g. by an HIV-infection), the contact with such vaccine strains can result in a salmonella infection.
Therefore, the object of the invention is to provide a live vaccine against salmonella infections, starting from the known prior art, being optimally attenuated for the host to be immunized, providing it with an immunity for reducing the excretion of wild strains when applied orally or parenterally, and constituting only a minor risk for people, especially those with a weakened immune system, or none at all. A further object of the invention is to provide a method for producing salmonella live vaccines optimally suited to the respective host by employing significantly less animal experiments than the conventional methods. Finally, the invention is to provide salmonella live vaccine strains for live vaccines suitable for chickens and poultry in general.
This object is attained by a specific live vaccine, a specific method for the production of salmonella live vaccines, and live vaccine strains.
The art described in this section is not intended to constitute an admission that any patent, publication or other information referred to herein is "prior art" with respect to this invention, unless specifically designated as such. In addition, this section should not be construed to mean that a search has been made or that no other pertinent information as defined in 37 C.F.R. .sctn. 1.56(a) exists.